Liquid Biopsy in Ovarian Cancer

Clin Chem Lab Med 2018; 56(2): 186–197

Review

Lydia Giannopoulou, Sabine Kasimir-Bauer and Evi S. Lianidou* Liquid biopsy in ovarian cancer: recent advances on circulating tumor cells and circulating tumor DNA https://doi.org/10.1515/cclm-2017-0019 by histological and molecular heterogeneity and is con- Received January 10, 2017; accepted March 2, 2017; previously sidered as a highly aggressive neoplasia. It is often diag- published online July 28, 2017 nosed at an advanced stage and little progress has been achieved in standard chemotherapy treatment and overall Abstract: Ovarian cancer remains the most lethal disease survival (OS) during the last 3 decades [2]. Primary disease among gynecological malignancies despite the plethora is treated with surgical removal of the tumor, followed by of research studies during the last decades. The major- standard adjuvant chemotherapy, a combination of plati- ity of patients are diagnosed in an advanced stage and num and taxane-based treatment [3, 4]. However, in more exhibit resistance to standard chemotherapy. Circulating than half of the cases, chemoresistance and recurrent tumor cells (CTCs) and circulating tumor DNA (ctDNA) disease are observed [5, 6]. New therapeutic concepts now represent the main liquid biopsy approaches that offer a include targeted therapy applying bevacizumab or the minimally invasive sample collection. Both have shown PARP inhibitor olaparib in certain clinical situations [7, 8]. a diagnostic, prognostic and predictive value in many Metastasis in ovarian cancer occurs via two main types of solid malignancies and recent studies attempted routes characterized by different molecular mechanisms, to shed light on their role in ovarian cancer. This review the transcoelomic passive dissemination of tumor sphe- is mainly focused on the clinical value of both CTCs and roids in the peritoneal fluid and ascites, and the hema- ctDNA in ovarian cancer and, more specifically, on their togenous metastasis of cancer cells in blood circulation potential as diagnostic, prognostic and predictive tumor and their preferred seeding to the omentum. Circulating biomarkers. tumor cells (CTCs) contribute to the hematogenous meta- Keywords: circulating tumor cells; circulating tumor DNA; static route [9, 10]. Generally, in solid malignancies, CTCs liquid biopsy; ovarian cancer; tumor biomarkers. are exceedingly rare, and in most cases, the amount of the available peripheral blood sample is limited. The development of different analytical systems for the detection, Introduction enumeration and molecular characterization of CTCs has expanded the field of liquid biopsy, providing information Ovarian cancer causes the majority of cancer-related on patients clinical outcome and treatment efficacy [11]. deaths from gynecological cancers and represents the Cell-free DNA (cfDNA) circulates at high concentra- third most frequent gynecological cancer worldwide [1]. tions in peripheral blood of cancer patients and can be Epithelial ovarian cancer is the main type, characterized used for the detection of several molecular alterations related to cancer development [12]. Circulating tumor DNA (ctDNA) represents a small percentage of cfDNA that *Corresponding author: Dr. Evi S. Lianidou, Analysis of Circulating is shed in circulation by tumor cells and carries all these Tumour Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, University Campus, Athens 15771, molecular alterations including tumor specific mutations, Greece, Phone: +30 210 7274311, Fax: +30 210 7274750, microsatellite instability (MI) [12], loss of heterozygosity E-mail: [email protected] (LOH) [13], and DNA methylation [14]. ctDNA is a very Lydia Giannopoulou: Analysis of Circulating Tumor Cells Lab, promising non-invasive diagnostic, prognostic and pre- Laboratory of Analytical Chemistry, Department of Chemistry, dictive tool, as it provides an easily accessible source of University of Athens, University Campus, Athens, Greece Sabine Kasimir-Bauer: Department of Gynecology and Obstetrics, DNA derived from the tumor [15]. University Hospital of Essen, University of Duisburg-Essen, Essen, In this review, we will give an overview of the published Germany data on CTCs and ctDNA in ovarian cancer (Figure 1). We

Unangemeldet Heruntergeladen am | 11.03.19 16:02 Giannopoulou et al.: Liquid biopsy in ovarian cancer 187

Plasma: ctDNA analysis

DNA mutations DNADNA mmethethylatioylation Ovarian cancer DNA integrity

Loss of heterozygosity DNANA ampllification/deletionifi i /d l / translocations

Point LOH mutations

CTCs detection and molecular characterization

CTCs enumeration DNA mutations DNA methylation

Simple blood draw Gene expression Loss of heterozygosity DNA amplification/deletion/ translocations

Point LOH mutations

Figure 1: CTCs and ctDNA in ovarian cancer. also focus on the possible role of liquid biopsy approaches and an immunocytochemical (ICC) assay [35], respectively. in early diagnosis, prognosis of clinical outcome and the Marth et al. [36] found carcinoma cells in the peripheral prediction of chemotherapy response or the development blood in 12% of ovarian cancer patients with a median of chemoresistance in ovarian cancer patients. follow-up of 25 months. The blood collection took place 7–20 days after surgery and before adjuvant chemotherapy. Judson et al. [35] detected CTCs in 18.7% of ovarian cancer Circulating tumor cells (CTCs) patients with 18.7 months of a median follow-up time. They observed that most women with CTCs had grade 3 Recent studies attempted to show the prognostic and pre- primary ovarian tumor compared to women without CTCs, dictive value of CTCs as tumor biomarkers in patients with and this evidence was significantly different. Both studies ovarian cancer [16], and three meta-analyses report these reported no significant association between the presence associations using the appropriate methods for the results of CTCs in the peripheral blood and the clinical outcome statistical analysis [17–19]. Different detection methods of ovarian cancer patients [35, 36]. were used, mainly based on immunocytochemistry Fan et al. [34] first reported the prognostic signifi- (microscopic detection or the FDA-approved CellSearch® cance of CTCs in primary ovarian cancer. They developed system), RT-PCR (AdnaTest, QIAGEN, Hilden, Germany), a new method for the detection of CTCs based on the and RT-qPCR for the quantification of CTCs levels [20, 21]. ability of cancer cells to invade and ingest a cell adhesion The time point of blood collection also differed, however, matrix (CAM). In this study, CTC detection was based on in the majority of studies the peripheral blood samples ICC using the epithelial markers epithelial cell adhesion were obtained before surgical removal of the tumor. An molecule (EpCAM), epithelial specific antigen (ESA) and overview of all research studies on CTCs in ovarian cancer a panel of seven pan-cytokeratins. They reported that the patients is presented in Table 1. CAM + CTCs were invasive and their presence significantly The first studies on CTCs in ovarian cancer were based correlated with decreased progression-free survival(PFS) on the detection of CTCs using specific immunobeads [36] (p = 0.042) [34]. The same group evaluated the prognostic

Unangemeldet Heruntergeladen am | 11.03.19 16:02 188 Giannopoulou et al.: Liquid biopsy in ovarian cancer NS NS p = 0.0024 NR NR PFS p = 0.001 (AC) NS p = 0.042 p < 0.00001 p = 0.0293 (AC) NS p = 0.0024 NR NS p = 0.009 NS NS p = 0.0017 NR NR OS p = 0.001 (AC) p = 0.0054 (BS) p = 0.047 (AC) NS NR p = 0.0008 (AC) NS p = 0.0219 NR NS p = 0.026 , EpCAM EpCAM, CK8,18,19 EpCAM, M30 CK8,18,19, EpCAM, CK8,18,19 12 gene panel including: including: panel 12 gene EpCAM , MUC1 MUC16 CK18 , 19 ERCC1 EpCAM , MUC1 MUC16 CK18 , 19 Targeted antigen/gene Targeted 12 gene panel including: including: panel 12 gene PPIC EpCAM , MUC1 HER2 CA125 EpCAM, ESA, CK4,5,6,8,10,13,18 EpCAM, ESA, CA125, DPP4 EpCAM, ESA, EpCAM , MUC1 MUC16 ERCC1 CK7,8,18,20, TFS-2, EGFR TFS-2,CK7,8,18,20, EGFR EpCAM, ESA, CA125, DPP4 EpCAM, ESA, Three multi-marker panels panels multi-marker Three stem and EMT epithelial, for cells associated transcipts MOC-31 MOC-31 EpCAM , MUC1 MUC16 ERCC1 ® ® ® CellSearch CellSearch CellSearch Cytomorphological/qPCR Cytomorphological/qPCR CTCs detection CTCs RT-qPCR (AdnaTest) RT-PCR ICC ICC AdnaTest Ovarian Ovarian AdnaTest CancerDetect/RT-PCR ICC ICC Multiplex-RT-PCR/ Ovarian AdnaTest CancerDetect Immunomagnetic beads Immunomagnetic RT-PCR (AdnaTest) RT-PCR ) ) ® ® ® ® ® CTCs isolation CTCs Density gradient gradient Density centrifugation AdnaTest Immunomagnetic CAM + method Size-based (MetaCell Size-based Immunomagnetic CAM + method AdnaTest Ovarian Ovarian AdnaTest CancerSelect CellSearch CellSearch Immunomagnetic microbeads Immunomagnetic Immunomagnetic CAM + method AdnaTest Ovarian Ovarian AdnaTest AdnaTest CancerSelect/ EMT-1/StemCellSelect Immunomagnetic Immunomagnetic (Dynabeads) AdnaTest Ovarian Ovarian AdnaTest CancerSelect Size-based (MetaCell Size-based CellSearch Ovarian cancer cancer Ovarian patients 216 122 58 118 (20 pts: gene gene 118 (20 pts: study) expression 123 (31 pts: study) monitoring 65 54 216 53 76 10 (3 pts: single single 10 (3 pts: cell analysis) 90 143 56 78 Sampling time Sampling Before surgery and after after and surgery Before chemotherapy and/or after surgery Before chemotherapy surgery Before Before surgery Before before surgery, Before a during and chemotherapy follow-up 24 months Before surgery (BS) and after after and (BS) surgery Before (AC) chemotherapy Before and after temsirolimus after and Before line chemotherapy first After surgery Before Before surgery Before Before surgery Before After surgery/before surgery/before After chemotherapy Before surgery Before Before surgery and and surgery Before longitudinally Serial measurements during during measurements Serial chemotherapy Year 2013 2011 2009 2015 2015 2016 2011 2011 2003 2014 2016 2002 2014 2016 2013 CTCs in ovarian cancer. in ovarian 1: CTCs Table Author Obermayr Obermayr [30] et al. Aktas [32] et al. et al. Fan [34] Kolostova Kolostova [25] et al. Pearl [26] et al. Chebouti Chebouti [22] et al. Behbakht Behbakht [31] et al. Poveda [33] et al. Judson [35] et al. Pearl [27] et al. Blassl [23] et al. Marth [36] et al. Kuhlmann Kuhlmann [28] et al. Kolostova Kolostova [24] et al. NS, no significance; NR, not reported; OS, overall surviaval; PFS, progression-free survival. PFS, progression-free surviaval; overall OS, reported; NR, not NS, no significance; Liu et al. Liu et al. [29]

Unangemeldet Heruntergeladen am | 11.03.19 16:02 Giannopoulou et al.: Liquid biopsy in ovarian cancer 189 significance of CTCs in a group of 129 pre-surgery ovarian overlapping fraction of ERCC1-expressing CTCs, which cancer patients using the same method for the detection is potentially selected by platinum-based chemotherapy. and identification of CTCs and observed statistically sig- Moreover, we describe that the assessment of CTC-derived nificant association between the presence of CTCs and ERCC1-transcripts alone is almost equivalently sufficient both OS (p = 0.0219) and PFS (p = 0.0024) [27]. The same in detecting ERCC1-expressing prognostic relevant CTCs. group also investigated the predictive value of CTC levels We further showed that the presence of ERCC1 + CTCs in a small group of 31 ovarian cancer patients that received after chemotherapy correlates with post-therapeutic standard taxol/carboplatin chemotherapy, where blood outcome of ovarian cancer and particularly, dynamics of specimens were obtained at different time points, before ERCC1 + CTCs mirror response to platinum-based chemo- and after surgery and up to 24 months after chemotherapy therapy [22]. treatment. Using the same assay [27], they showed a sta- Poveda et al. [33] also confirmed the prognostic impact tistically significant association between CTC levels and of CTC detection in ovarian cancer after chemotherapy. disease progression [26]. They reported a correlation of CTC numbers with shorter Aktas et al. investigated the prognostic value of CTCs OS (p = 0.0017) and PFS (p = 0.0024) in a phase III clinical in a large cohort of 122 ovarian cancer patients, before trial (NCT00113607, www.clinicaltrials.gov) of pegylated surgery and/or after platinum-based chemotherapy. They liposomal doxorubicin (PLD) with trabectedin versus PLD used the commercially available AdnaTest BreastCancer for relapsed ovarian cancer. They used for the first time (QIAGEN, Hilden, Germany), for the isolation and detec- the CellSearch® system (Janssen Diagnostics) for CTC iso- tion of CTCs. AdnaTest BreastCancer is based on immuno- lation and enumeration in 216 ovarian cancer patients. magnetic enrichment targeting EpCAM and anti-mucin 1 Behbakht et al. also used the CellSearch® system for CTC (MUC1), followed by multiplex RT-PCR for EpCAM, MUC1 enrichment and enumeration in a phase II clinical trial and human growth factor receptor 2 (HER2/neu). CA-125 (NCT00429793, www.clinicaltrials.gov) for the evaluation transcripts were also analyzed using a singleplex RT-PCR. of the efficacy of the mTOR inhibitor temsirolimus. Fifty CTCs were detected in 19% of patients before surgery and four recurrent ovarian cancer patients were recruited and in 27% after platinum-based chemotherapy. According blood specimens were obtained before and after treatment to their findings, the presence of CTCs significantly cor- with temsirolimus. No significant association between the related with shorter OS before surgery (p = 0.0054) and presence of CTCs with PFS and OS was reported [31]. Liu after chemotherapy (p = 0.047) [32]. In a more recent et al. [29] also used the CellSearch® system in 78 newly study, Kuhlmann et al. investigated the predictive value of diagnosed and recurrent ovarian cancer patients. They ERCC1-positive CTCs in 143 pre-surgery epithelial ovarian performed serial measurements during chemotherapy, cancer patients. AdnaTest OvarianCancerSelect (QIAGEN, but according to their findings, the number of CTCs did Hilden, Germany) was used for the immunomagnetic not correlate with PFS or OS. tumor cell enrichment in blood samples and AdnaT- Obermayr et al. [37] developed a six-marker gene est OvarianCancerDetect (QIAGEN, Hilden, Germany) panel for the molecular detection of CTCs on female cancer for the molecular characterization of CTCs. ERCC1 tran- patients, including ovarian cancer, using a RT-qPCR plat- script detection was performed using singleplex RT-PCR. form. The multimarker analysis using this novel panel The presence of CTCs was confirmed in 14% of patients positively identified 19% of the 23 ovarian cancer patients. and was significantly correlated with OS (p = 0.041). The same group aimed to identify novel markers for the ERCC1-positive CTCs (ERCC1 + CTC) were detected in 8% characterization of CTCs in ovarian cancer, using a density of patients and significantly correlated with both OS gradient centrifugation-based method for the isolation (p = 0.026) and PFS (p = 0.009). A very interesting finding and RT-qPCR for CTC detection and quantification. They in this study was the association of ERCC1 + CTC with plat- defined a sample as CTC positive if at least one of the 11 inum resistance. The presence of ERCC1 + CTC at primary gene marker panels was found over-expressed. By using diagnosis independently predicted platinum resistance this gene panel, they detected CTCs in 24.3% of the base- (p = 0.010), although the ICC analysis of ERCC1 expres- line (before primary treatment) and 20.4% of the follow- sion in primary tumor tissue did not reveal any prognostic up (6 months after chemotherapy) samples. In two-thirds or predictive value [28]. In their very recently published of the patients, cyclophilin C gene (PPIC) overexpression study, they were able to show that the additional assess- was observed, but only a few samples were identified by ment of ERCC1-transcripts enhances overall CTC detec- EpCAM overexpression. PPIC-positive CTCs during follow- tion rate in ovarian cancer patients before surgery and up were detected significantly more often in platinum- after chemotherapy and defines an additional highly resistant than platinum-sensitive follow-up patients. This

Unangemeldet Heruntergeladen am | 11.03.19 16:02 190 Giannopoulou et al.: Liquid biopsy in ovarian cancer fact also indicated poor outcome independently from Cell-free DNA (cfDNA) other prognostic parameters [30]. Kolostova et al. [38] developed a novel size-based A sufficient number of studies on cfDNA in patients with method (MetaCell®, MetaCell s.r.o., Ostrava, Czech ovarian cancer pursued to clarify its clinical value [39]. Republic) for the enrichment and separation of viable For this purpose, they quantified total cfDNA and/or the CTCs, followed by in vitro CTCs culturing and cytomorpho- circulating cell-free mitochondrial DNA (mtDNA) levels logical analysis and finally, CTC molecular characterization in some cases, or aimed at the detection of different by gene expression studies using qPCR. They isolated and genetic and epigenetic alterations, such as chromosomal cultivated CTCs in 77 (65.2%) of 118 pre-surgery advanced- abnormalities and specific tumor LOH, cancer-related stage ovarian cancer patients. Gene expression analysis somatic gene mutations and aberrant DNA methylation. was performed in 20 selected positive samples by cytomor- Additionally, in a recent case study, Martignetti et al. phological analysis. They looked at possible associations [40] detected the FGFR2-FAM76A tumor-specific fusion between CTC presence and clinicopathological character- in cfDNA of an advanced stage serous epithelial ovarian istics of the patients, mainly with the CA-125 status. Based cancer patient. on their results, they proposed a new and independent However, in some cases, the results are still contro- prognosis staging information. They also suggest that versial. The discrepancies probably occur due to the dif- hematogenous metastasis route is represented by CTCs ferent methods and pre-analytical conditions, the use and elevated CA-125 levels indicate lymphogenic dissemi- of serum instead of plasma by some researchers and the nation [25]. Using the same methodology, this group aimed different volumes of plasma/serum for cfDNA extrac- to isolate and identify CTCs in 56 ovarian cancer patients. tion. Many studies focused on the potential use of cfDNA In this study, gene expression analysis was performed in as a diagnostic, prognostic and predictive biomarker in all samples found positive by cytomorphological analysis. ovarian cancer and a recent meta-analysis by Zhou et al. They reported that EpCAM relative expression is elevated attempted to evaluate the role of cfDNA in ovarian cancer in CTC-enriched fractions compared to whole peripheral diagnosis [41]. An overview of the research studies on blood sample and that this expression grows with in vitro cfDNA in ovarian cancer is summarized in Table 2. cultivation time. They suggested that a seven-gene panel, The first studies on ovarian cancer circulating DNA including EpCAM and MUC16, could better confirm the attempted to quantify the total cfDNA amount, or the presence of CTCs in peripheral blood of ovarian cancer nuclear and mitochondrial DNA amounts separately, in patients, than a one-marker test [24]. Both studies did not plasma or serum of ovarian cancer patients. One of the provide any information on the patients clinical outcome first studies on cfDNA in ovarian cancer screening aimed with regard to OS and/or PFS data [24, 25]. to quantify plasma cfDNA using a real-time PCR assay for A very recent study on CTCs in ovarian cancer pro- three reference genes and to determine the number of posed a multi-marker gene panel for gene expression pro- genome equivalents (GE) using a standard curve. Kamat filing of single CTCs [23]. Blassl et al. used the AdnaTest et al. [42] reported that cfDNA levels in advanced ovarian OvarianCancerSelect (QIAGEN, Hilden, Germany) and/ cancer samples were elevated when compared to controls. or the AdnaTest EMT-1/StemCellSelect (QIAGEN, Hilden, A more recent study on ovarian cancer screening using Germany) for CTC isolation and enrichment in peripheral cfDNA quantification showed a significant increase in blood samples of 10 pre-surgery epithelial ovarian cancer serum cfDNA of advanced stage ovarian cancer patients patients. CTCs were detected and characterized by using compared to early stage (p < 0.01). Shao et al. [47] also the AdnaTest OvarianCancerDetect (QIAGEN, Hilden, reported a correlation between serum cfDNA levels and Germany) and the AdnaTest EMT-1/StemCellDetect. They ovarian cancer occurrence using receiver operating char- isolated single cells using CellCelector (ALS GmbH, Jena, acteristic (ROC) curves and a branched DNA (bDNA) tech- Germany) from only three ovarian cancer patients. Single nique for cfDNA quantification. CTCs were characterized by multiplex-RT-PCR, followed Kamat et al. also investigated the prognostic value by capillary electrophoresis. The multiplex-RT-PCR gene of cfDNA in epithelial ovarian cancer. They quantified panel included stem cell (CD44, ALDH1A1, Nanog, Oct 4) plasma cfDNA levels in 164 epithelial ovarian cancer and EMT (N-cadherin, Vimentin, Snail2, CD117, CD146) patients using real-time PCR for β-globin and determined markers. They observed inter-cellular and intra/inter- the number of GE. They reported a significant association patient heterogeneity and co-expression of epithelial, of cfDNA > 22,000 GE/mL with decreased PFS (p < 0.001) mesenchymal and stem cell transcripts on the same CTC and this association was shown as an independent prog- simultaneously [23]. nostic value (p = 0.02) after adjusting for other clinical

Unangemeldet Heruntergeladen am | 11.03.19 16:02 Giannopoulou et al.: Liquid biopsy in ovarian cancer 191 Yes Yes Yes Yes NR Yes Yes Response to to Response treatment Yes Yes Yes Yes NR Yes Yes Yes Yes Prognosis Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes NR Yes Yes Yes Early Early detection , , CES4A , EP300 PGR- , BRCA1 , CDKN1C , CDH1 RUNX3 , RASSF1A SLIT2 APC , OPCML , SFRP5 TFPI2 RASSF2A OPCML , OPCML RUNX3 , TFPI2 RASSF1A BRCA1 , RASSF1A , , PGR-PROX BRCA1 , HIC1 PAX5 THBS1 , CALCA RASSF1A PROX RASSF1A hMLH1 Panels including: TP53 , PIK3CA including: Panels BRAF , EGFR TP53 , PIK3CA including: Panels , KRAS BRAF FBXW7 MET , PTEN , BRAF KRAS , EGFR TP53 , PTEN PIK3CA RB1 , ZEB2 MTOR BUB1 , PARP8 KRAS TP53 TP53 - - - - MTATP8 MTATP8 NR (bDNA technique) Cyclophilin A Cyclophilin B2M , RAB25 CLDN4 ABCF2 , β - actin GADPH , β - actin globin GADPH hTERT Targeted gene Targeted 36 87 47 45 114 59 50 33 30 106 138 7 22 46 3 126 27 69 32 57 10 63 21 100 (20 follow-up) 36 144 36 164 19 22 Ovarian cancer cancer Ovarian patients Serum Serum Plasma Serum Serum Plasma Plasma/serum Plasma Plasma Serum Plasma Plasma Serum Plasma Plasma Plasma Plasma Plasma/serum Plasma Plasma Plasma Serum Plasma/serum Plasma Serum Plasma Serum Plasma Plasma Plasma Source Year 2012 2013 2014 2014 2015 2017 2004 2009 2011 2011 2004 2014 2015 2012 2013 2011 2004 2005 2016 2016 2016 2012 2008 2014 2015 2014 2012 2010 2006 2008 Zhang et al. [67] et al. Zhang [68] et al. Wu [69] et al. Zhou [70] et al. Wang Dong et al. [66] Dong et al. Giannopoulou et al. [65] et al. Giannopoulou Melnikov et al. [62] et al. Melnikov [63] et al. Liggett [64] et al. Bondurant Ibanez et al. [61] et al. Ibanez Gifford et al. [60] et al. Gifford Bettegowda et al. [58] et al. Bettegowda [59] et al. Pereira Murtaza et al. [57] et al. Murtaza Forshew et al. [56] et al. Forshew Dobrzycka et al. [55] et al. Dobrzycka Swisher et al. [54] et al. Swisher Otsuka et al. [53] et al. Otsuka Cohen et al. [51] et al. Cohen [52] et al. Vanderstichele Harris et al. [50] et al. Harris Kuhlmann et al. [13] et al. Kuhlmann Choudhuri et al. [49] et al. Choudhuri Zachariah et al. [48] et al. Zachariah Shao et al. [47] et al. Shao Steffensen et al. [46] et al. Steffensen No et al. [45] No et al. Kamat et al. [44] et al. Kamat Capizzi et al. [43] et al. Capizzi Kamat et al. [42] et al. Kamat Author Aberrant methylation methylation Aberrant (ctDNA) Somatic mutations (ctDNA) mutations Somatic Chromosomal Chromosomal (ctDNA) abnormalities/LOH Mitochondrial (mtDNA) Mitochondrial DNA amount (cfDNA) DNA amount cfDNA cfDNA in ovarian cancer. in ovarian 2: cfDNA Table

Unangemeldet Heruntergeladen am | 11.03.19 16:02 192 Giannopoulou et al.: Liquid biopsy in ovarian cancer characteristics [44]. On the contrary, No et al. [45] exam- levels of the follow-up patients before and after treatment, ined the prognostic value of cfDNA and reported no but not in circulating cell-free mtDNA levels [49]. significant difference between cfDNA levels of cancer patients and patients with benign disease. They recruited 36 epithelial ovarian cancer samples and 16 benign tumor samples and used commercially available copy number Circulating tumor DNA (ctDNA) assay kits to measure cfDNA levels of four selected genes, but they used serum as cfDNA source instead of plasma. Circulating tumor DNA (ctDNA) constitutes a tiny sub- In a more recent study, Steffensen et al. measured group of total cfDNA in the peripheral blood of cancer plasma cfDNA levels of 144 multiresistant epithelial patients [73]. The following studies refer on specific aber- ovarian cancer patients treated with bevacizumab using rations characterizing ctDNA shed in the circulation from real-time PCR for cyclophiline A gene. They found a sta- the primary ovarian tumor. They are classified according tistically significant correlation between cfDNA levels and to specific genetic or epigenetic alterations detected only both PFS (p = 0.0004) and OS (p = 0.005) in both univariate in ctDNA, shown as below. and multivariate survival analyses. Thus, they concluded that plasma cfDNA is an independent prognostic factor in platinum-resistant ovarian cancer patients treated with Chromosomal abnormalities/LOH bevacizumab [46]. Ten years ago, Kamat et al. [71] proposed the potential It is well known that ovarian cancer and in particular the use of tumor-specific cfDNA levels in predicting tumor high-grade serous ovarian cancer (HGSC) subtype, is char- response to chemotherapy, by using an orthotopic mouse acterized by frequent chromosomal instability [5]. Recent model. Capizzi et al. further investigated the predictive studies aimed to detect copy number variations (CNV) value of cfDNA in ovarian cancer patients. They quanti- [51] and to quantify specific LOH [13] or aberrant somatic fied plasma cfDNA levels before and after chemotherapy chromosomal rearrangements [50] in ctDNA of ovarian in 22 epithelial ovarian cancer patients of a prospective cancer patients. Kuhlmann et al. quantified cfDNA of 63 nonrandomized clinical study and found a significant dis- primary epithelial ovarian cancer patients before surgery crimination between patients and healthy controls and a and after chemotherapy. They used a PCR-based fluores- correlation of cfDNA amounts with response to standard cence microsatellite analysis in order to measure the LOH chemotherapy [43]. in two fractions of cfDNA, the high- and low molecular- Altered circulating cell-free mtDNA content may serve weight fraction (HMWF and LMWF, respectively). They as a potential cancer biomarker in many solid malig- reported that LOH at two markers can predict tumor grade nancies [72]. In ovarian cancer, only two studies include (p = 0.033) and FIGO stage (p = 0.004) in the LMWF cfDNA. the determination of circulating cell-free mtDNA levels. Remarkably, a LOH at another marker can significantly Zachariah et al. quantified nuclear cfDNA and circulat- predict patients OS (p = 0.030) in both HMWF and LMWF ing cell-free mtDNA levels using a multiplex qPCR assay, [13]. in serum and plasma of patients with epithelial ovarian Harris et al. introduced an algorithm for the quanti- cancer, benign epithelial tumors and endometriosis, and fication of cfDNA using a qPCR assay in order to predict a healthy control group. They found a significant increase relapse and treatment efficacy. They identified aberrant in nuclear cfDNA and circulating cell-free mtDNA amounts chromosomal junctions in primary tumors of 10 ovarian in ovarian cancer patients compared to both healthy cancer patients and detected them in plasma ctDNA of group and benign epithelial tumor patients. Interestingly, eight patients before surgery. In three cases, ctDNA was they reported a significant difference between ovarian also detected after surgery, indicating the presence of the cancer patients and the endometriosis group circulat- disease, but in the remaining five cases, ctDNA was absent ing cell-free mtDNA, but not in nuclear cfDNA [48]. More after surgery, indicating the consequential absence of the recently, Choudhuri et al. investigated whether nuclear disease [50]. cfDNA and circulating cell-free mtDNA levels can be used The first study on ovarian cancer screening using for advanced epithelial ovarian cancer diagnosis and for CNV detection in cfDNA was elaborated by Cohen et al. the prediction of treatment response. They recruited 100 [51]. They applied a well-established non-invasive pre- patients and measured both levels before treatment, but natal testing (NIPT) commercial platform in cfDNA of 16 in only 20 patients after the completion of chemotherapy. pre-surgery early and 16 advanced HGSC patients. The A significant difference was reported in nuclear cfDNA obtained sequencing data were analyzed for the detection

Unangemeldet Heruntergeladen am | 11.03.19 16:02 Giannopoulou et al.: Liquid biopsy in ovarian cancer 193 of subchromosomal changes and the determination of Murtaza et al. performed whole exome sequencing whole chromosome gains or losses. They detected 40.6% in plasma ctDNA of three ovarian cancer patients. Serial of all HGSC cases, and more specifically, 38% of early sample measurements and quantification of allele frac- stages, indicating a potential utility for early HGSC screen- tions in ctDNA led to the identification of specific gene ing in plasma cfDNA based on specific multiple segmental mutations related to acquired resistance to treatment. The chromosome gains and losses [51]. However, more valida- genes with significantly increased mutant AFs are shown tion studies along with the improvement of pre-analytical in Table 2. All results were confirmed using both digital conditions and the examination of paired tumor DNA are PCR and Tam-Seq assay [57]. needed before the routine application of this approach [74]. Another study by Bettegowda et al. accomplished Vanderstichele et al. reported for the first time the the detection of ctDNA using digital PCR-based assays potential of using cfDNA for primary HGSC diagnosis. for mutation analyses in a large cohort of patients with They recruited 68 patients with an adnexal mass, includ- different malignancies, including seven patients with ing 57 diagnosed with invasive or borderline carcinoma advanced stage ovarian cancer. They detected ctDNA and 11 with benign disease. They measured specific pat- in most metastatic cancer patients and quantified the terns of chromosomal instability in plasma cfDNA of all mutant fragments for the determination of cfDNA con- patients and reported a significantly higher quantitative centration. They reported a high mutant allele fragments measure of chromosomal instability in ovarian cancer (approximately 10,000 per 5 mL) for advanced ovarian patients compared to patients with benign disease or cancer patients [58]. healthy individuals [52]. In a more recent study, Pereira et al. recruited patients with gynecological malignancies, including 22 ovarian patients, and identified specific cancer-related muta- Somatic mutations tions using whole exome and targeted sequencing. They also measured and quantified ctDNA levels using droplet Few studies attempted to detect tumor-specific somatic digital PCR (ddPCR). The detectable ctDNA after treatment mutations in ctDNA of epithelial ovarian cancer patients. significantly predicted survival for eight ovarian cancer Otsuka et al. [53] first identified TP53 mutations in patients, indicating a possible role of ctDNA measure- only two/12 pre-surgery plasma cfDNA of patients with ments in personalized medicine [59]. ovarian cancer. A tumor-specific TP53 mutation was also detected in 21 out of 69 cfDNA samples of epithelial ovarian cancer patients in a study by Swisher et al. Aberrant methylation The presence of ctDNA characterized by this mutation was significantly associated with decreased survival Epigenetic alterations hold an important role in cancer (p = 0.02) [54]. Mutations of KRAS gene were investi- initiation and progression and aberrant DNA methylation gated by Dobrzycka et al. in plasma cfDNA of 126 epi- patterns, mainly characterized by promoter hypermeth- thelial ovarian cancer patients. They detected KRAS ylation, are a frequent event in most human cancers [75]. mutations in 43.7% of patients and reported a signifi- Epigenetic inactivation of a tumor suppressor gene often cantly decreased OS for patients with serous ovarian results from its promoter methylation and is considered tumors and detectable cfDNA (p = 0.022) [55]. as an early event during carcinogenesis [76]. Many studies The development of very sensitive novel technologies have reported methylation changes in ovarian cancer [77] for ctDNA detection overcomes the issue of the extremely and a recent review summarizes the differences in the low concentrations of ctDNA out of the total cfDNA. Based on observed methylation patterns in the main histological this concept, Forshew et al. proposed a different approach subtypes of the disease, including HGSC [78]. DNA meth- for the detection and identification of cancer-specific muta- ylation changes have the potential to serve as biomarkers tions in plasma ctDNA. They established a novel method for for early diagnosis of gynecological malignancies [79]. targeted deep sequencing (Tam-Seq) of mutations at low This is also observed in Table 2; only one study by Gifford allele frequencies (AF) with increased sensitivity and speci- et al. [60] aimed to show the prognostic value of ctDNA ficity, and measured mainly the frequencies of TP53 mutant methylation in ovarian cancer. alleles at ctDNA of 46 advanced stage HGSC patients. In this study, the researchers investigated hMLH1 Remarkably, an EGFR mutation was detected in one ctDNA methylation status in plasma cfDNA of 138 epithelial sample but not in the initial ovarian tumor tissue. All results ovarian cancer patients enrolled in a phase III clini- were confirmed using digital PCR [56]. cal trial (NCT00003998, www.clinicaltrials.gov), before

Unangemeldet Heruntergeladen am | 11.03.19 16:02 194 Giannopoulou et al.: Liquid biopsy in ovarian cancer carboplatin/taxoid chemotherapy and at relapse. They cfDNA methylation levels were significantly increased reported an increase in hMLH1 methylation at relapse and in ovarian cancer patients compared to benign disease the remarkable presence of cfDNA methylation at 25% of patients and healthy control groups [70]. relapse patients that was not detected before chemother- Furthermore, studies on SLIT2 [66], OPCML [69] and apy. This acquired methylation provided significant clini- RASSF2A [68] promoter methylation in cfDNA of epithelial cal information for patients OS (p = 0.007) [60]. ovarian cancer patients demonstrate the frequently aber- Ibanez et al. examined RASSF1A and BRCA1 hyper- rant methylation status of these genes and suggest a pos- methylation in cfDNA of 50 epithelial ovarian cancer sible role for ovarian cancer early detection. patients and first confirmed the detection of methyla- Methylation patterns in whole-blood DNA and tion in early stage (stage I, II) patients, using methylation white blood cell (WBC) DNA in ovarian cancer patients specific PCR (MSP). They also observed a concordance have been also examined using methylation arrays and between tumor and plasma/serum DNA methylation pat- bisulfite pyrosequencing. Teschendorff et al. [80] per- terns in 82% of matched samples [61]. formed a methylation study in peripheral blood DNA of A microarray mediated methylation assay (MethDet pre- and post-treatment ovarian cancer patients and they test) was developed by Melnikov et al. [62] and its applica- observed a significantly different methylation pattern tion in 33 serous ovarian cancer patients led to the charac- in blood DNA of epithelial ovarian cancer patients com- terization of a five genes panel for ovarian cancer detection. pared to healthy controls. Flanagan et al. [81] investi- The same group used this assay in three cohorts of serous gated WBCs DNA methylation status in 880 epithelial ovarian cancer patients, benign ovarian disease patients ovarian cancer patients enrolled in a phase III clinical trial and healthy controls. Liggett et al. [63] now reported the (NCT00003998, www.clinicaltrials.gov), using bisulfite distinctive promoter methylation of all three groups accord- pyrosequencing and reported a significant correlation ing to the methylation status of six selected genes. between mean SFN methylation and PFS (p = 0.016). The A larger study by Bondurant et al. quantified RASSF1A same group analyzed blood DNA methylation patterns in promoter methylation in 106 serous ovarian cancer cfDNA 247 ovarian cancer patients enrolled in the previous clini- samples, using a novel quantitative real-time PCR assay. cal trial. They identified specific CpGs alterations in blood They found RASSF1A promoter methylation in about DNA at relapse after platinum-based chemotherapy and half of ovarian cancer patients and observed agreement found an independent significant association with sur- in the methylation status of 20 available paired tumor/ vival (p = 2.8 × 10−4) [82]. serum samples. Interestingly, they measured RASSF1A methylation in nine patients over the course of treatment and found a concordance between cfDNA methylation changes and disease progression for eight patients, sug- Conclusions gesting a possible role of cfDNA methylation in ovarian cancer prognosis [64]. The development of a cancer biomarker and its imple- Our group also reported RASSF1A promoter methyla- mentation in the clinical routine requires a multistage tion in plasma ctDNA of 15/59 patients with high-grade procedure and constitutes the final result of multiannual serous ovarian cancer using a real-time MSP assay. We and toilsome research approaches. However, multiple pre- performed the first comparison study on RASSF1A pro- analytical, analytical and post-analytical issues should be moter methylation in primary tumors, adjacent tissues overcome and studies on the assay validations with regard and plasma samples in HGSC patients and we observed to repeatability and reproducibility are also necessary an agreement between primary tumor samples and corre- [83]. The lack of effective biomarkers for early detection, sponding plasma in 62.3% of cases studied [65]. prognosis of clinical outcome and response to treatment Zhang et al. developed a multiplex-MSP assay for the contributes to the maintenance of low survival rates for early detection of ovarian cancer. They recruited 87 epi- ovarian cancer patients, despite the numerous research thelial ovarian cancer patients and examined the serum studies on the field, the last decades. Liquid biopsy pro- cfDNA methylation status of seven selected genes simul- cedures are minimally invasive and allow for the easily taneously. A sample was characterized as positive, if at tolerated serial sample measurements during the course least one gene was found methylated [67]. In a more recent of treatment. This can help towards the establishment study by Wang et al., a multiplex-nested MSP was also of more efficient personalized therapeutic algorithms developed for the detection of three genes methylation and real-time therapy monitoring. Nevertheless, specific in 114 serum cfDNA of epithelial ovarian cancer patients. challenges should be taken into account for CTCs and

Unangemeldet Heruntergeladen am | 11.03.19 16:02 Giannopoulou et al.: Liquid biopsy in ovarian cancer 195 ctDNA analyses, including pre-analytical issues about the 8. Ledermann J, Harter P, Gourley C, Friedlander M, Vergote I, sample volume, the proper tubes for sample collection, Rustin G, et al. Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a pre- the samples storage and the time of the analysis, quality planned retrospective analysis of outcomes by BRCA status in a control and analytical validation of the assays. randomised phase 2 trial. Lancet Oncol 2014;15:852–61. The clinical significance of both CTCs and ctDNA has 9. Tan DS, Agarwal R, Kaye SB. Mechanisms of transcoelomic been revealed in many types of cancer [84], including metastasis in ovarian cancer. Lancet Oncol 2006;7:925–34. ovarian cancer. However, no standard methods are used 10. 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Kuhlmann JD, Schwarzenbach H, Wimberger P, Poetsch M, responsibility for the entire content of this submitted Kimmig R, Kasimir-Bauer S. LOH at 6q and 10q in fractionated manuscript and approved submission. circulating DNA of ovarian cancer patients is predictive for tumor Research funding: None declared. cell spread and overall survival. BMC Cancer 2012;12:325. Employment or leadership: None declared. 14. Warton K, Samimi G. Methylation of cell-free circulating DNA in the diagnosis of cancer. Front Mol Biosci 2015;2:13. Honorarium: None declared. 15. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic Competing interests: The funding organization(s) played acids as biomarkers in cancer patients. Nat Rev Cancer no role in the study design; in the collection, analysis, and 2011;11:426–37. interpretation of data; in the writing of the report; or in the 16. Romero-Laorden N, Olmos D, Fehm T, Garcia-Donas J, Diaz- decision to submit the report for publication. Padilla I. 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